DE102014109687B4 - Position determination of an object in the beam path of an optical device - Google Patents

Abstract

A method for determining a position (150) of an object (100) parallel to the optical axis (120) of an optical device (1), the method comprising: - illuminating the object (100) from a first illumination direction (210-1) and detecting a first image (230-1) of the object (100) during the illumination, - illuminating the object (100) from a second illumination direction (210-2) and capturing a second image (230-2) of the object (100) during the Illuminating, - determining a distance (250) between imaging locations (220-1, 220-2) of the object (100) in the first image (230-1) and in the second image (230-2), - determining a position ( 150) of the object (100) parallel to the optical axis (120) based on the distance (250), the first illumination direction (210-1) being characterized by a first angle (251-1) with respect to the optical axis (120), the second direction of illumination (210-2) by a second angle (251-2) with respect to the optical axis (120), wherein the determination of the position (150) of the object (100) is further based on the first angle (251-1) and on the second angle (251-2).

Description

Various aspects relate to a method for determining a position of an object parallel to the optical axis of an optical device and a corresponding device.

The determination of the position of an object to be imaged parallel to the optical axis of an optical device (z position) can be desirable for various reasons. Thus, by means of the known z position, it can be possible to position the object as well as possible within the focal plane of the optical device in order to be able to produce a particularly sharp image of the object in this way. In the case of objects extended perpendicular to the optical axis, it may be desirable to determine the z position for different points of the object perpendicular to the optical axis in order to be able to focus on the relevant image section. It may also be desirable to create a height profile of the object using optical techniques.

Existing techniques allow the z position to be determined e.g. by positioning the object at different reference positions. A sharpness of the object at the various reference positions can then be used to assess whether the object is in the focal plane or not. However, it can often only be possible with a limited accuracy to determine a sharpness of the object. Therefore, such a prior art technique can be comparatively inaccurate. Interferometric techniques for determining the z position are also known. Such techniques allow a comparatively high accuracy when determining the z position; the corresponding devices can, however, be comparatively complicated and expensive.

Therefore, there is a need for improved techniques for determining a position of an object parallel to the optical axis of an optical device. In particular, there is a need for such techniques that are comparatively easy to implement in optical devices, i.e. require little or no structural changes, and which allow a comparatively high accuracy of determining the position.

This task is solved by the independent claims. The dependent claims define embodiments.

In one aspect, the present application relates to a method for determining a position of an object parallel to the optical axis of an optical device. The method includes illuminating the object from a first illumination direction and capturing a first image of the object during the illumination. The method further comprises illuminating the object from a second illumination direction and capturing a second image of the object during the illumination. The method further includes determining a distance between imaging locations of the object in the first image and the second image. The method further includes determining a position of the object parallel to the optical axis based on the distance.

In other words, it may be possible to illuminate the object sequentially from the first and second illumination directions and to capture an image of the object in each case. In particular, the first illumination direction and / or the second illumination direction can enclose an angle with that axis of the optical device along which an idealized light beam experiences no or only a slight deflection (optical axis). In such a case, the imaging location of the object can be shifted in a corresponding image, provided that the object is not in the focal plane of the optical device. By determining the distance in relation to the first and second direction of illumination, it may be possible to draw conclusions about the position.

Determining the position can mean: quantitative determination of the position, e.g. in relation to the focal plane or in relation to another suitable reference system of the optical device; and / or determining the position qualitatively, e.g. with regard to the criterion whether a certain predetermined position is parallel to the optical axis, e.g. the focus level, is reached or not.

The first direction of illumination is characterized by a first angle with respect to the optical axis and the second direction of illumination is characterized by a second angle with respect to the optical axis. Determining the position of the object continues to be based on the first angle and the second angle. In such a case, it can in particular be possible that the determination of the position of the object further comprises: quantifying the position of the object with respect to the focal plane of the optical device on the basis of trigonometric relationships between the angles and the distance.

With such an approach, it may be possible, for example, to carry out a comparatively precise determination of the position of the object parallel to the optical axis based solely on the first and second image. In particular, it can be unnecessary, for example, to carry out a series of different images of the object for different reference positions of the object parallel to the optical axis. In other words, it may be possible Determine the position of the object only on the basis of the images for different lighting directions; it may not be necessary to move the object mechanically parallel to the optical axis. This can enable the position to be determined particularly quickly and precisely. This can enable a particularly simple implementation of the corresponding measurement process. For example, in comparison to conventional optical devices, such as microscopy devices, it may be possible to change only one illumination device of the optical device; For example, it may be possible for a detection device of the optical device to remain unchanged.

The present application further includes a method for determining a position of an object parallel to the optical axis of an optical device. The method includes: illuminating the object from a first illumination direction and capturing a first image of the object during the illumination, illuminating the object from a second illumination direction and capturing a second image of the object during the illumination, determining a distance between imaging locations of the object in the first Image and in the second image, determining a position of the object parallel to the optical axis based on the distance, and positioning the object at different reference positions parallel to the optical axis. The method can e.g. for each of the reference positions parallel to the optical axis include illuminating the object from the first illumination direction and capturing the first image and illuminating the object from the second illumination direction and capturing the second image and determining the distance. Determining the position of the object can then include: minimizing the distance for the different reference positions. In such a case, it may in particular be possible to qualitatively determine the position of the object parallel to the optical axis. For example, in the event that the distance becomes minimal, it can be assumed that the corresponding reference position is parallel to the optical axis in or close to the focal plane. E.g. two, three or more reference positions can be used. In principle, it is also possible to adapt the directions of illumination as a function of the reference position. Provision can also be made for capturing more than two images from more than two illumination directions for the different reference positions. In this way, multiple imaging locations can be determined or redundant information can be obtained, which enables a particularly precise position determination. Particularly in scenarios in which the object has a certain periodicity or is a periodic structure, a particularly precise determination of the position can be possible in this way.

In general it may be possible e.g. following the determination of the position of the object to control a focus unit of the optical device, for positioning the object in the focus plane of the optical device as a function of the determined position. In this way it can be possible to focus on the object in a particularly fast, reliable and precise manner. Subsequently, images of the object can be captured that have a particularly high quality.

Various techniques can be used to determine the distance. For example, determining the distance may include determining a first reference point of the object in the first image and determining a second reference point in the second image. The distance can be determined between the first reference point and the second reference point. The first and second reference points can correspond to a certain part of the object. The distance can be determined particularly precisely by a suitable choice of the reference point.

In general, the choice of a suitable reference point is not particularly limited. In particular, it may be desirable to choose a reference point that can be found and determined both in the first image and in the second image with a comparatively high reliability and accuracy. Then an accuracy of the determined position can be comparatively high. The reference points would be e.g. in question: distinctive features of the object; Landmarks; machine readable characters; points set by a user, etc.

If the object has a significant extent perpendicular to the optical axis, the choice of the reference point can have an influence on the part of the object for which the position is determined. This can be particularly important in scenarios in which the object has a significant height profile. A scenario can then occur in which the focusing of one part of the object goes hand in hand with the defocusing of another part of the object. In such a case, it may be desirable to create a so-called focus map, i.e. e.g. to determine spatially resolved information about the position of the object perpendicular to the optical axis.

For example, the distance can be determined for several pairs of first reference points and second reference points. The position of the object can be determined in a location-resolved manner in a plane perpendicular to the optical axis and based on the plurality of pairs of first reference points and second reference points. In this way it can be possible, for example to selectively position individual parts of the object in a focal plane of the optical device. This can be particularly desirable in the case of samples extended perpendicular to the optical axis.

Typically, an accuracy of determining the position of the object parallel to or along the optical axis correlates with an accuracy of determining the distance between the imaging locations of the object in the first image and the second image. This means that it may be desirable to determine the distance between the imaging locations particularly precisely. For example, the distance can be determined using techniques selected from the following group: landmark recognition; Determining an optical center of gravity of the object in the first image and / or the second image; a user input; Aberration correction.

For example, it may be possible to take into account known aberrations e.g. in the illumination device of the optical device and / or in the detector optics of the optical device to take into account distortions in the first and second images, which can lead to a shift in the imaging locations of the object. Such shifts can then be calculated out or reduced mathematically and the actual distance can be determined particularly precisely.

According to a further aspect, the present application relates to an optical device. The optical device is set up to determine a position of an object parallel to or along the optical axis of the optical device. The optical device comprises a lighting device. The lighting device is set up to illuminate the object from a first lighting direction and from a second lighting direction. The optical device further comprises a detector which is set up to acquire a first image of the object during the illumination from the first direction of illumination. The detector is also set up to acquire a second image of the object during the illumination from the second direction of illumination. The optical device further comprises a computing unit which is set up to determine a distance between imaging locations of the object in the first image and in the second image. The computing unit is also set up to determine a position of the object parallel to the optical axis based on the distance, the first illumination direction being characterized by a first angle with respect to the optical axis, the second illumination direction being characterized by a second angle with respect to the optical axis and the computing unit is set up to further determine the position of the object based on the first angle and on the second angle.

The present application further includes an optical device that is configured to determine a position of an object parallel to the optical axis of the optical device. The optical device comprises an illumination device, a detector and a computing unit. The lighting device is set up to illuminate the object from a first lighting direction and from a second lighting direction. The detector is set up to capture a first image of the object during the illumination from the first illumination direction and to capture a second image of the object during the illumination from the second illumination direction. The computing unit is set up to determine a distance between imaging locations of the object in the first image and in the second image and to determine a position of the object parallel to the optical axis based on the distance. The computing unit is also set up to control a sample holder for positioning the object at different reference positions parallel to the optical axis. The optical device is set up for each reference position parallel to the optical axis in order to carry out the illumination of the object from the first illumination direction and the acquisition of the first image and the illumination of the object from the second illumination direction and the acquisition of the second image and the determination of the propriety. The computing unit is set up to minimize the distance for the different reference positions when determining the position of the object.

For example, the optical device in accordance with the aspect currently being discussed may be configured to perform the method for determining a position of an object parallel to the optical axis in accordance with another aspect of the present application.

For such an optical device, effects can be achieved that are comparable to the effects that can be achieved for the method for determining a position of an object parallel to the optical axis according to a further aspect of the present application.

The features and features set out above, which are described below, can be used not only in the corresponding explicitly stated combinations, but also in further combinations or in isolation, without departing from the scope of protection of the present invention.

• 1 illustriert eine Position eines Objekts parallel zur optischen Achse einer optischen Vorrichtung.
• 2 illustriert Abbildungsorte des Objekts in einem ersten Bild und in einem zweiten Bild, die für verschiedene Beleuchtungsrichtungen aufgenommen sind, für das Szenario der 1.
• 3 zeigt schematisch die optische Vorrichtung.
• 4 ist ein Flussdiagramm eines Verfahrens zum Bestimmen der Position des Objekts parallel zur optischen Achse.
• 5 zeigt das iterative Positionieren des Objekts an verschiedenen Referenzpositionen parallel zur optischen Achse, um die Position qualitativ zu bestimmen.
• 6 illustriert eine Abbildung eines Objekt in ersten und zweiten Bildern, wobei das Objekt senkrecht zur optischen Achse ausgedehnt ist, wobei Referenzpunkte zur Bestimmung des Abstands dargestellt sind.

The above-described properties, features and advantages of this invention and the manner in which they are achieved can be more clearly understood in connection with the following description of the exemplary embodiments, which are explained in more detail in connection with the drawings.

• 1 illustrates a position of an object parallel to the optical axis of an optical device.
• 2nd illustrates locations of the object in a first image and in a second image, which are recorded for different lighting directions, for the scenario of FIG 1 .
• 3rd shows schematically the optical device.
• 4th is a flowchart of a method for determining the position of the object parallel to the optical axis.
• 5 shows the iterative positioning of the object at different reference positions parallel to the optical axis in order to determine the position qualitatively.
• 6 illustrates an image of an object in first and second images, the object being extended perpendicular to the optical axis, with reference points for determining the distance being shown.

The present invention is explained in more detail below on the basis of preferred embodiments with reference to the drawings. In the figures, the same reference symbols designate the same or similar elements. The figures are schematic representations of various embodiments of the invention. Elements shown in the figures are not necessarily drawn to scale. Rather, the various elements shown in the figures are reproduced in such a way that their function and general purpose can be understood by the person skilled in the art. Connections and couplings between functional units and elements shown in the figures can also be implemented as an indirect connection or coupling. A connection or coupling can be implemented wired or wireless. Functional units can be implemented as hardware, software or a combination of hardware and software.

Techniques are described below by means of which a position of an object parallel to the optical axis of an optical device (z position) can be determined. In the three-dimensional space spanned by x, y, z axes, the z component of the position can be determined; the optical axis defines the z-axis and is e.g. parallel to this. Based on the determined z position, e.g. a focus unit of the optical device can be controlled and in this way the object can be positioned in the focus plane of the optical device as a function of the determined z position (focusing of the object). Subsequently, images of the object can be captured which depict the object particularly sharply. Such techniques can be used in a wide variety of fields, e.g. in microscopy or in fluorescence measurement or in parallel to phase contrast imaging.

For the exemplary application of fluorescence measurement, it can e.g. be possible to determine the z position before and / or during the fluorescence measurement using the techniques described below. This can ensure that the fluorescent object is in the focal plane of the optical device during the measurement; in this way, an accuracy in the fluorescence measurement can be increased. The techniques described in detail below are based on evaluating a first image and a second image while illuminating the object from different first and second illumination directions. The lighting can e.g. in particular with a wavelength that lies outside the fluorescence-active region of the fluorescent sample. Basically, the z position can be determined simultaneously with the fluorescence measurement. This can e.g. in particular, enable moving samples to be reliably positioned in the focus plane as a function of time. Furthermore, the z position can generally be determined from only two lighting processes; this can also reduce a light-toxic effect on the fluorescent object. When measuring dyes, the wavelength of the light can be used to determine the z position e.g. can be selected outside the range of excitation of the dyes. In this way, bleaching of the dyes can be reduced or avoided. A possible wavelength of light that is used to determine the z position would be e.g. in the infrared range.

In particular, images from different directions of illumination can already be present in different scenarios, without these having to be recorded especially for the focusing according to the present techniques. Such a scenario would be e.g. the determination of a phase contrast image using techniques of Fourier ptychography. Then, without further exposure of the object to light, it may be possible to use the techniques at hand to determine the z position.

In 1 is an optical device 1 , for example a microscope, shown schematically. A ray path of light runs from one Lighting device 111 to a detector 112 . In 1 are the optical axis 120 and the focus level 160 shown. Out 1 it can be seen that the object 100 parallel to the optical axis 120 out of focus 160 is positioned. A z position is shown 150 that are related to the focus level 160 is measured (in 1 With Δz designated). In such a case, the focus unit of the optical device can be particularly easily and quickly possible 1 to control to perform a focus. In particular, it can be unnecessary, for example converting the z position 150 in terms of the focus level 160 perform. It would also be possible to position the object 100 to be determined in another suitable reference coordinate system of the optical device.

In 2nd are also a first direction of illumination 210-1 and a second direction of illumination 210-2 shown. For the first lighting direction 210-1 becomes a first picture 230-1 detected. For the second lighting direction 210-2 becomes a second picture 230-2 detected. How from 2nd the first direction of illumination closes 210-1 a first angle 251-1 with the optical axis 120 on. Therefore an image location appears 220-1 of the object 100 in the first picture 230-1 according to 2nd to the left opposite the optical axis 120 transferred. In 2nd is the first angle 251-1 as α designated. How from 2nd is also visible is the location of the picture 220-2 of the object 100 in the second picture 230-2 in the representation of the 2nd to the right opposite the optical axis 120 transferred. This is because of the second angle 251-2 (in 2nd With β designated) that the second direction of illumination 210-2 with the optical axis 120 includes. Out 2nd it can be seen that an amount of the first angle 251-1 different from an amount of the second angle 251-2 is. In general, it would be possible for the first and second directions of illumination 210-1 , 210-2 symmetrical with respect to the optical axis 120 are arranged. For example, it would also be possible for one of the two directions of illumination 210-1 , 210-2 parallel to the optical axis 120 is oriented. In general, it is also possible that the object 100 an offset from the optical axis 120 has, ie within an xy plane with respect to the optical axis 120 is moved. In general, it is also not necessary that the first direction of illumination 210-1 , the second direction of illumination 210-2 and the optical axis 120 lie in one level (in the scenario of 2nd in the xz plane). This means that, for example, the first direction of illumination 210-1 and / or the second direction of illumination 210-2 may have tilted out of the xy plane.

Because lighting the object 100 with finite angles 251-1 , 251-2 opposite the optical axis 120 takes place, a pure phase object, which causes no or only a slight weakening of the amplitude of the light passing through, can also occur in the first and second images 230-1 , 230-2 be mapped. This enables the present techniques to be used in a variety of ways on different samples, in particular, for example, biological samples.

In 2nd is also a distance 250 between the locations 220-1 , 220-2 of the object 100 in the first and second pictures 230-1 , 230-2 shown (in 2nd With Δx designated). First of all, it can be determined qualitatively that the distance 250 does not disappear. So can the z position 150 can already be determined qualitatively as non-zero. For example, it would be possible to reposition the object iteratively 100 at different reference positions (in 2nd not shown) parallel to the optical axis 120 who have favourited Z Position 150 to be determined qualitatively as equal or close to zero. For this purpose, the object could be iteratively parallel to the optical axis 120 be repositioned until the distance 250 is minimized.

But it would also be possible to determine the z position 150 continue on the first angle 251-1 and the second angle 251-2 based. Then the z position 150 be determined quantitatively. For this purpose, as explained below, trigonometric relationships between the first angle 251-1 , the second angle 251-2 and the distance 250 be taken into account.

wobei a einen Abstand zwischen dem Objekt 100 und dem Abbildungsort 220-1 des Objekts 100 in dem ersten Bild 230-1 entlang der ersten Beleuchtungsrichtung 210-1 bezeichnet und b einen Abstand zwischen dem Objekt 100 und dem Abbildungsort 220-2 des Objekts 100 in dem zweiten Bild 230-2 entlang der zweiten Beleuchtungsrichtung 210-2 bezeichnet (a und b sind in 2 nicht dargestellt). Diese Formel ergibt sich aus der Definition des Cosinus für rechtwinklige Dreiecke.For the scenario of 2nd applies: $$\Delta \mathrm{e.g.}=a\cdot \text{cos}\alpha =b\cdot \text{cos}\beta ,$$

where a is a distance between the object 100 and the location of the image 220-1 of the object 100 in the first picture 230-1 along the first direction of illumination 210-1 denotes and b a distance between the object 100 and the location of the image 220-2 of the object 100 in the second picture 230-2 along the second direction of illumination 210-2 (a and b are in 2nd not shown). This formula results from the definition of the cosine for right triangles.

Using the sine theorem for general triangles we get: $$\frac{\Delta x}{\text{sin}\left(\alpha +\beta \right)}=\frac{b}{\text{sin}\left(90°-\alpha \right)}=\frac{b}{\text{cos}\alpha }.$$

Combining equations 1 and 2 gives: $$\Delta \mathrm{e.g.}=\Delta x\cdot \frac{\text{cos}\alpha \text{cos}\beta }{\text{sin}\left(\alpha +\beta \right)}.$$

Using equation 3, it is possible based on the first angle 251-1 and the second angle 251-2 and further based on the distance 250 of the locations 220-1 , 220-2 who have favourited Z Position 150 to determine. In particular, the z position 150 simply by illuminating twice and simultaneously capturing the first and second image 230-1 , 230-2 be determined. A light exposure of the object 100 can be minimized, e.g. in comparison to the above scenario with iterative positioning of the object 100 at different reference positions parallel to the optical axis 120 .

It may be desirable to have an accuracy of determining the z position 150 to increase. The accuracy of determining the z position 150 typically depends directly on the first angle 251-1 , the second angle 251-2 and the distance 250 together. Therefore, the accuracy in determining the z position 150 be limited at least by a pixel size in the first image 230-1 and the second picture 230-2 .

A mistake in the distance 250 - hereinafter as Δx ' designated - is transferred to an error in z position 150 as follows: $$\Delta \mathrm{e.g.}\text{'}=\Delta x\text{'}\cdot \frac{\text{cos}\alpha \text{cos}\beta }{\text{sin}\left(\alpha +\beta \right)}.$$

Unless the object 100 has a significant extent in the xy-plane, it may be desirable, for example, the distance 250 between certain reference points in the first image 230-1 and the second picture 230-2 to determine. The reference points can be a specific part of the object 100 mark, for example a particularly striking part or a part that is particularly important for the illustration. In general, it is also possible to change the distance 250 for several pairs of reference points of the object 100 to determine. In this way, it can be possible to apply equation 3 several times for different parts of the object 100 each the z position 150 to determine. In other words, the z position 150 be determined in a spatially resolved manner in the xy plane.

So it may be worth striving for the distance 250 to be determined particularly precisely. In this context, it may be possible to use a wide variety of techniques to determine the distance particularly precisely 250 enable. Such techniques can include, for example: landmark recognition; Determination of an optical center of gravity of the object 100 in the first picture 230-1 and / or in the second picture 230-2 ; a user input; an aberration correction. In a simple scenario, for example, the user could determine a specific reference point of the object 100 in the first picture 230-1 select and the corresponding reference point in the second image 230-2 choose. Using landmark recognition, it may be possible, for example, to carry out such a selection of reference points at least partially automatically. It would also be possible to use the optical center of gravity as a reference point to determine the distance 250 to use. The aberration correction can be used, for example, for known malformations due to aberrations in the optical device 1 to consider.

Another limitation of accuracy in determining the z position 150 can result from the coherent depth of field of the detector 112 the optical device 1 surrender. In particular, it should be ensured that the object 100 - Even if there is a significant shift compared to the focus level 160 - still in the first picture 230-1 and the second picture 230-2 is mapped. However, it may not be necessary to have a sharp image of the object 100 to reach; in particular the techniques described above, such as determining the optical center of gravity of the object 100 , can also be used in a case where the object 100 only very blurred in the pictures 230-1 , 230-2 is shown.

While in the 1 and 2nd a situation is shown in which the object 100 along the optical axis 120 is positioned, ie it is the optical axis 120 intersects, using the techniques described above, the z position can also be determined for those scenarios in which the object 100 a certain offset parallel to the x direction and / or parallel to the y direction with respect to the optical axis 120 having. In general terms, the techniques described above can be used to determine position 150 of the object parallel to the optical axis 120 thus the determination of the z component of the position of the object 100 in three-dimensional space spanned by the x, y, z axes.

In the 2nd a situation is also shown in which two directions of illumination 210-1 , 210-2 can be used to determine the z position. In general, it is also possible to use a larger number of lighting directions 210-1 , 210-2 to determine the z position 150 of the object 100 to use. For example, three or four or ten or more lighting directions 210-1 , 210-2 be used. It would be possible, for example, for the different lighting directions 210-1 , 210-2 the above techniques in pairs to apply, eg to apply equation 3 in pairs. For example, the z position 150 of the object 100 be determined several times and a suitable mean value can be formed from them. For example, the z position may be possible in this way 150 of the object 100 to be determined particularly precisely. In general, a variety of techniques can be used to combine a larger data set consisting of mapping locations 220-1 , 220-2 multiple lighting directions 210-1 , 210-2 be used. For example, equation 3 could be appropriately modified or multiple z positions arising from the different directions of illumination 210-1 , 210-2 could be consolidated after applying Equation 3 several times. In other words, it can be through multiple lighting directions 210-1 , 210-2 or redundant lighting directions 210-1 , 210-be possible, a higher accuracy when determining the z position 150 to reach; for example, it is possible, in particular, to achieve an accuracy which is higher than a resolution of corresponding images from which the imaging locations 220-1 , 220-2 be determined.

In 3rd is the optical device 1 shown schematically. The optical device 1 has the lighting device 111 and the detector 112 on. There is also a sample holder with focus unit 311 intended. The focus unit can be set up to the object 100 parallel to the optical axis 120 position, for example to approach or focus on different reference positions. The optical device 1 also has a computing unit 312 on. The computing unit 312 is set up various steps related to determining the z position 150 , as explained above. The computing unit 312 can with a memory (in 3rd not shown) coupled. In the memory, for example a non-volatile or volatile memory, corresponding work instructions and commands for carrying out the above techniques by the computing unit 312 be deposited. For example, the computing unit 312 Receive commands from memory to use equation 3 to determine the z position 150 to determine or within the first and second image 230-1 , 230-2 Find reference points and then the distance 250 to investigate.

In general, it is possible to use the optical device 1 other tasks - in addition to determining the z position 150 - Are carried out, such as fluorescence measurements. In such a case, in particular, the z position can be determined 150 based on the first and second images 230-1 , 230-2 be carried out by means of auxiliary optics which, for example, have a small aperture with a large depth of field - which can ensure that even for large z positions 150 the distance 250 can still be reliably determined. Additional optics can then be used to carry out the actual fluorescence measurement, which, for example, has a large diaphragm in order to work particularly light-intensively. Also parallel acquisition of the first and second images 230-1 , 230-2 and performing the fluorescence measurement may be possible in this way.

Basically, a wide variety of lighting devices 111 used to illuminate the object 100 with the different lighting directions 210-1 , 210-2 perform. For example, a scan mirror can be used, for example, in a field diaphragm plane of the lighting device 111 be used. An adaptive component could also be used in an aperture diaphragm or illumination pupil of the illumination device; for example, the lighting device 111 according to German patent application 10 2014 101 219.4 can be used. An adaptive component could be, for example, a spatial light modulator (SLM) or a digital micromirror device (DMD) or a movable or displaceable sigma diaphragm. It would also be possible for the lighting device 111 comprises a light-emitting diode array. For example, the light emitting diodes of the light emitting diode array can be arranged on a Cartesian grid. Then, for example, by driving a certain light-emitting diode of the light-emitting diode array, which is a certain distance from the optical axis 120 has a certain direction of illumination 210-1 , 210-2 be implemented.

In 4th is a method of determining the z position 150 of the object 100 shown according to various embodiments. The process begins in step S1 . First in step S2 the object 100 from the first lighting direction 210-1 illuminated and the first picture 230-1 detected. In step S3 becomes the object 100 from the second lighting direction 210-2 illuminated and the second picture 230-2 detected. Then in step S4 the distance 250 between the two locations of the object in the first and second images 230-1 , 230-2 certainly. Then takes place in step S5 determining the z position 150 . In step S5 can determine the z position 150 for example qualitatively or quantitatively. For quantitative determination of the z position 150 For example, equation 3 can be used. It would also be possible that - in addition to the steps S2 - S4 - The object from other lighting directions 210-1 , 210-2 is illuminated, for example from a third direction of illumination and a fourth direction of illumination. This redundant information can be found in step S5 be taken into account.

But it would also be possible to step in S5 the z position 150 through iterative repositioning of the object 100 parallel to the optical axis 120 to determine qualitatively. Such a scenario is in 5 shown. First in step T1 the distance 250 of the object 100 between the first picture 230-1 and the second picture 230-2 for a current reference position of the object 100 parallel to the optical axis 120 certainly. In step T2 it checks whether the distance 250 is minimized. For example, in step T2 a threshold value comparison can be carried out with a predetermined threshold value. It would be in step T2 also possible to check whether the distance 250 compared to previous determinations of distance (for previous iterations of the step T1 ) was reduced.

Was in step T2 found that the distance 250 has not been minimized, so step T3 carried out. In step T3 becomes the object 100 at a next reference position parallel to the optical axis 120 positioned. The reference positions can be determined using an iterative process; the reference positions could also be predefined. The process then goes to step S2 continued (cf. 4th ). In particular for a determination of the z position 150 using iterative techniques as in 5 shown, it may be desirable to have more than two directions of illumination 210-1 , 210-2 to determine the distance 250 to use. For example, in step T2 be checked whether the distance 250 for all pairs of lighting directions 210-1 , 210-2 is minimized.

Will in step T3 however found that the distance 250 is minimized, so in step T4 the z position 150 as zero compared to the focus plane 160 certainly.

In 6 is an illustration of an object 100 in the first picture 230-1 (in 6 shown with a solid line) and in the second picture 230-2 (in 6 with a dashed line) is shown schematically. The object 100 has a significant extent in the xy plane, ie perpendicular to the optical axis 120 having. There are three possible reference points 600-1 , 600-2 , 600-3 , 600-4 for the illustration of the object 100 in the first picture 230-1 shown. Basically the choice of reference points 600-1 - 600-4 not particularly limited. In the 6 shown reference points 600-1 - 600-4 but can be particularly reliable in the first and second picture 230-1 , 230-1 be found. For example, the distance 250 between the first reference point 600-1 be determined (cf. 6 ), because this is the highest point of the object 100 in the pictures 230-1 , 230-2 and thus easy and reliable to find. The reference point 600-4 denotes, for example, the optical focus of the object 100 in the pictures 230-1 , 230-2 .

In summary, techniques have been described above which - for example by applying equation 3 or by repositioning the object parallel to the optical axis - determine the z position particularly quickly and precisely 150 enable. A quick focus on the object 100 this becomes possible.

Of course, the features of the previously described embodiments and aspects of the invention can be combined with one another. In particular, the features can be used not only in the combinations described, but also in other combinations or on their own without leaving the field of the invention.

For example, techniques have been described above in which the object is illuminated from two directions of illumination. This can be particularly advantageous if exposure of the object to light is to be minimized. However, it would generally also be possible to use a larger number of lighting directions, e.g. if it is necessary to determine the position of the object particularly parallel to the optical axis.

Furthermore, mainly scenarios were discussed above in relation to the figures, in which essentially the entire object is focused. In general, however, it may be possible to focus only one relevant image section that depicts only part of the object, or to determine the z position of the relevant part of the object.

Furthermore, mainly those scenarios were discussed above in which the object is positioned perpendicular to the optical axis in such a way that it intersects the optical axis. However, it would also be possible for the object to be offset with respect to the optical axis.

Claims (10)

A method for determining a position (150) of an object (100) parallel to the optical axis (120) of an optical device (1), the method comprising: - illuminating the object (100) from a first illumination direction (210-1) and detecting a first image (230-1) of the object (100) during the illumination, - illuminating the object (100) from a second illumination direction (210-2) and capturing a second image (230-2) of the object (100) during the Lighting, - Determining a distance (250) between imaging locations (220-1, 220-2) of the object (100) in the first image (230-1) and in the second image (230-2), - determining a position (150) of the Object (100) parallel to the optical axis (120) based on the distance (250), the first illumination direction (210-1) being characterized by a first angle (251-1) with respect to the optical axis (120), the second Illumination direction (210-2) is characterized by a second angle (251-2) with respect to the optical axis (120), the determination of the position (150) of the object (100) still on the first angle (251-1) and on the second angle (251-2). Procedure according to Claim 1 wherein determining the position (150) of the object (100) further comprises: - quantifying the position (150) of the object (100) with respect to the focal plane (160) of the optical device (1) based on trigonometric relationships between the first Angle (251-1), the second angle (251-2) and the distance (250). A method for determining a position (150) of an object (100) parallel to the optical axis (120) of an optical device (1), the method comprising: Illuminating the object (100) from a first illumination direction (210-1) and capturing a first image (230-1) of the object (100) during the illumination, Illuminating the object (100) from a second illumination direction (210-2) and capturing a second image (230-2) of the object (100) during the illumination, - determining a distance (250) between imaging locations (220-1, 220-2) of the object (100) in the first image (230-1) and in the second image (230-2), - Determining a position (150) of the object (100) parallel to the optical axis (120) based on the distance (250), - Positioning the object (100) at different reference positions parallel to the optical axis (120), the method for each reference position parallel to the optical axis (120) illuminating the object (100) from the first illumination direction (210-1) and detecting it of the first image (230-1) and illuminating the object (100) from the second illumination direction (210-2) and capturing the second image (230-2) and determining the distance (250), wherein determining the position (150) of the object (100) comprises: - Minimize the distance (250) for the different reference positions. A method according to any one of the preceding claims, the method further comprising: - Controlling a focus unit (311) of the optical device (1) for positioning the object (100) in the focus plane (160) of the optical device (1) as a function of the determined position (150). The method of any one of the preceding claims, wherein determining the distance (250) comprises: - determining a first reference point (600-1 - 600-4) of the object (100) in the first image (230-1), - Determining a second reference point (600-1 - 600-4) in the second image (230-2), the distance (250) between the first reference point (600-1 - 600-4) and the second reference point (600- 1 - 600-4) is determined. Procedure according to Claim 5 , wherein the distance (250) is determined for a plurality of pairs of first reference points and second reference points, the position (150) of the object (100) being spatially resolved in a plane perpendicular to the optical axis (120) based on the plurality of pairs of first reference points and second reference points. Method according to one of the preceding claims, wherein the distance (250) is determined by means of techniques selected from the following group: landmark recognition; Determining an optical center of gravity of the object (100) in the first image (230-1) and / or the second image (230-2); a user input; Aberration correction. Optical device (1) which is set up to determine a position (150) of an object (100) parallel to the optical axis (120) of the optical device (1), the optical device (1) comprising: - an illumination device ( 111), which is configured to illuminate the object (100) from a first illumination direction (210-1) and from a second illumination direction (210-2), - a detector (112), which is configured to acquire a first image (230-1) of the object (100) during the illumination from the first illumination direction (210-1) and a second image (230-2) of the object (100) during the illumination from the second illumination direction (210-2) a computer unit (312) which is set up to determine a distance (250) between imaging locations (220-1, 220-2) of the object (100) in the first image (230-1) and in the second image (230-2) and determine a position (150) of the object (100) parallel to the optical axis (120) based on f to determine the distance (250), the first illumination direction (210-1) being characterized by a first angle (251-1) with respect to the optical axis (120), wherein the second direction of illumination (210-2) is characterized by a second angle (251-2) with respect to the optical axis (120), the computing unit (312) being set up to further base the position (150) of the object (100) on the first angle (251-1) and on the second angle (251-2). Optical device (1) which is set up to determine a position (150) of an object (100) parallel to the optical axis (120) of the optical device (1), the optical device (1) comprising: an illumination device (111) which is set up to illuminate the object (100) from a first illumination direction (210-1) and from a second illumination direction (210-2), - A detector (112) which is set up to detect a first image (230-1) of the object (100) during illumination from the first illumination direction (210-1) and a second image (230-2) of the object (100) during the illumination from the second illumination direction (210-2), - a computing unit (312) which is set up to calculate a distance (250) between imaging locations (220-1, 220-2) of the object (100) in the first image (230-1) and in the second image (230- 2) and to determine a position (150) of the object (100) parallel to the optical axis (120) based on the distance (250), wherein the computing unit (312) is further set up to control a sample holder (311) for positioning the object (100) at different reference positions parallel to the optical axis (120), wherein the optical device (1) is set up for each reference position parallel to the optical axis (120) to illuminate the object (100) from the first illumination direction (210-1) and capture the first image (230-1) and that Illuminating the object (100) from the second illumination direction (210-2) and capturing the second image (230-2) and determining the distance (250), wherein the computing unit is set up to minimize the distance (250) for the different reference positions when determining the position (150) of the object (100). Optical device after Claim 8 or 9 , wherein the optical device (1) is set up to carry out a method according to any one of claims 1-7.

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